Obligate intracellular bacterial pathogens are significant causes of human disease. Two major hindrances to studying these organisms are (i) systems for efficiently transforming them are lacking and (ii) inactivating a gene that is criticalfor invasion or intracellular survival prevents recovery of the mutant. As a result, much of their pathobiology and bona fide roles of their putative virulence factors remain unclear. We will develop a system that achieves high transformation efficiency of obligate intracellular bacteria while inside host cells, effectively targets pathogen-occupied vacuoles, and enables interference of expression of specific genes to elucidate their functions. Poly(amido)amine (PAMAM) dendrimers are highly branched macromolecules that are used for gene therapy and drug delivery into mammalian cells. Dendrimers can be functionally modified to enhance their transformation efficiency, targeting to specific cell types, and specific cellular trafficking pathways. We will functionally modify dendrimers to target pathogen-occupied vacuoles of obligate intracellular bacteria. Anaplasma phagocytophilum is an obligate intracellular bacterium that causes the potentially deadly disease, human granulocytic anaplasmosis. We determined that dendrimers would deliver plasmid DNA into the A. phagocytophilum-occupied vacuole (ApV) to transform the bacterium. However, the efficiency was poor, suggesting that dendrimers need to be functionally modified to effectively target the ApV. We discovered that ApV intercepts anterograde sphingolipid-rich vesicles from the trans-Golgi network (TGN). Exogenously added ceramide (a sphingolipid) accumulates in the TGN, from there traffics into ~90% of ApVs, and is then directly incorporated by Ap. We hypothesize that covalently linking dendrimers to ceramide will dramatically increase their trafficking to the ApV and uptake by A. phagocytophilum. This approach has great potential to improve transformation of not only Ap, but also other obligate intracellular bacteria that hijack TGN-derived sphingolipids. Indeed, chlamydiae are known to parasitize the TGN, and herein we extend this phenomenon to Ehrlichia chaffeensis. In Aim 1, we will functionally modify dendrimers to improve transformation efficiency of A. phagocytophilum, E. chaffeensis, and Chlamydia spp. In Aim 2, we will use dendrimers to achieve gene- specific knock down in A. phagocytophilum. Completing these aims will yield an easy to use, highly efficient and modifiable system for transforming and enabling gene-targeting studies in obligate intracellular bacteria. Moreover, this system could potentially be tailored to specifically target pathogen-occupied vacuoles as a new generation of therapeutics against obligate intracellular pathogens.